Parallel Acquisition of Plasma Membrane Ultrastructure and Cytosolic Protein Localisation in Cultured Cells via Correlated Immunogold SEM

Isabell Begemann; Ulrike Keller; Harald Nüsse; Jürgen Klingauf; Milos Galic

doi:10.3390/cells9061329

Parallel Acquisition of Plasma Membrane Ultrastructure and Cytosolic Protein Localisation in Cultured Cells via Correlated Immunogold SEM

Cells. 2020 May 26;9(6):1329. doi: 10.3390/cells9061329.

Authors

Isabell Begemann^{1

2}, Ulrike Keller¹, Harald Nüsse¹, Jürgen Klingauf^{1

2}, Milos Galic^{1

2}

Affiliations

¹ Institute of Medical Physics and Biophysics, University of Muenster, Robert-Koch-Str. 31, 48149 Münster, Germany.
² Interfaculty Centre 'Cells in Motion', University of Muenster, Waldeyerstr. 15, 48149 Münster, Germany.

Abstract

Scanning electron microscopy (SEM) takes advantage of distinct detectors to visualise secondary and back-scattering electrons. Here, we report an integrated approach that relies on these two detection methods to simultaneously acquire correlated information on plasma membrane topography and curvature-sensitive cytosolic protein localization in intact cell samples. We further provide detailed preparation and staining protocols, as well as a thorough example-based discussion for imaging optimisation. Collectively, the presented method enables rapid and precise analysis of cytosolic proteins adjacent to cellular membranes with a resolution of ~100 nm, without time-consuming preparations or errors induced by sequential visualisation present in fluorescence-based correlative approaches.

Keywords: BAR domain; CLEM; SE/BSE; curvature-sensitive protein; immunogold labelling; scanning electron microscopy.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Membrane / ultrastructure*
Cells, Cultured
Cytosol / metabolism*
Gold / chemistry
Humans
Immunohistochemistry
Microscopy, Electron, Scanning*
Protein Transport
Reproducibility of Results

Substances

Gold